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Background:  New therapeutic strategies for HIV/AIDS are needed. Genetic engineering of hematopoietic stem cells (HSC) coupled with nonmyeloablative conditioning proved safe and effective in the treatment of ADA-deficient SCID. The feasibility of such an approach in HIV-1 infection is unknown.

Methods:  In a 48-week open-label prospective trial, 18 patients with HIV-1 infection (mean ± SE age 36 ± 1, range 19 to 40; HAART since ≥ 3 months; CD4⁺ > 200 cells/μL) have been enrolled in a HSC retroviral vector gene therapy trial using RevM10 and polAS as anti-HIV genes. Of the total, 9 patients received fresh transduced CD34⁺ cells and all study phases, including CD34⁺ cell mobilization with G-CSF (10 μg/kg/day for 5 days), CD34⁺ cell collection via apheresis, and nonmyeloablative conditioning (1.8 g/m² cyclophosphamide [CY]), while 9 did not undergo all study phases.

Results:  Mean ± SE baseline CD4⁺ T-cell counts were 577 ± 42, while plasma viral load was < 80 copies/mL (limit of Nasba Organon assay) in 9 of 18 patients. CD34⁺ cells were efficiently mobilized and collected from patients, achieving 4.42 ± 0.64 x 10⁶ CD34⁺ cells/kg after purification, and 3.93 ± 1.2x10⁶ viable CD34⁺ cells/kg in the infusion product, 30% of which were transduced CD34⁺ cells. Of note, effective viral load suppression significantly increased the yields of mobilization, purification, and transduction processes; and peripheral blood CD34⁺ cell counts before apheresis (mean, 72 cells/μL) predicted the number of viable CD34⁺ cells infused (β 0.722, 95% CI 0.007 to 0.092, p = 0.028, regression analysis), and a cut-off value > 30 CD34⁺ cells/μL predicted the success of all procedures (p = 0.018, χ² analysis, Fisher’s exact test). Gene marking levels were detectable in all patients, though decreasing over time. CY conditioning caused a marked decrease in CD4⁺ T-cell counts, restored over long-term follow-up. This recovery correlated with levels of CD4⁺ TCR-rearrangement excision circles and CD4⁺CD45RA⁺CCR7⁺ naïve T cells. Notably, CMV-specific IL-2- and IFN-γ-secreting CD4⁺CD69⁺ T cells were able to expand while no CMV reactivation occurred (DNA stably negative); moreover, proportions of IL-2, IL-2/IFN-γ, and IFN-γ-secreting TT, FLU, HSV, and EBV-specific CD4⁺ and/or CD8⁺ T cells were not altered by CY over time.

Conclusions:  These data indicate that effective stem cell gene transfer is feasible in patients with HIV-1 infection, and suggest the use of non-lymphocyte-toxic conditioning regimen, such as busulfan.

Keywords: HIV gene therapy; stem cell gene therapy; nonmyeloablative conditioning